Abstract
The abnormal proliferation of cancer cells is driven by deregulated oncogenes or tumor suppressors, among which the cancer-vulnerable genes are attractive therapeutic targets. Targeting mislocalization of oncogenes and tumor suppressors resulting from aberrant nuclear export is effective for inhibiting growth transformation of cancer cells. We performed a clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) screening in a unique model of matched primary and oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed cells and identified genes that were growth promoting and growth suppressive for both types of cells, among which exportin XPO1 was demonstrated to be critical for the survival of transformed cells. Using XPO1 inhibitor KPT-8602 and by small interfering RNA (siRNA) knockdown, we confirmed the essential role of XPO1 in cell proliferation and growth transformation of KSHV-transformed cells and in cell lines of other cancers, including gastric cancer and liver cancer. XPO1 inhibition induced cell cycle arrest through p53 activation, but the mechanisms of p53 activation differed among the different types of cancer cells. p53 activation depended on the formation of promyelocytic leukemia (PML) nuclear bodies in gastric cancer and liver cancer cells. Mechanistically, XPO1 inhibition induced relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. Taken the data together, we have identified novel growth-promoting and growth-suppressive genes of primary and cancer cells and have demonstrated that XPO1 is a vulnerable target of cancer cells. XPO1 inhibition induces cell arrest through a novel PML- and p62-dependent mechanism of p53 activation in some types of cancer cells.IMPORTANCE Using a model of oncogenic virus KSHV-driven cellular transformation of primary cells, we have performed a genome-wide CRISPR-Cas9 screening to identify vulnerable genes of cancer cells. This screening is unique in that this virus-induced oncogenesis model does not depend on any cellular genetic alterations and has matched primary and KSHV-transformed cells, which are not available for similar screenings in other types of cancer. We have identified genes that are both growth promoting and growth suppressive in primary and transformed cells, some of which could represent novel proto-oncogenes and tumor suppressors. In particular, we have demonstrated that the exportin XPO1 is a critical factor for the survival of transformed cells. Using a XPO1 inhibitor (KPT-8602) and siRNA-mediated knockdown, we have confirmed the essential role of XPO1 in cell proliferation and in growth transformation of KSHV-transformed cells, as well as of gastric and liver cancer cells. XPO1 inhibition induces cell cycle arrest by activating p53, but the mechanisms of p53 activation differed among different types of cancer cells. p53 activation is dependent on the formation of PML nuclear bodies in gastric and liver cancer cells. Mechanistically, XPO1 inhibition induces relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. These results illustrate that XPO1 is a vulnerable target of cancer cells and reveal a novel mechanism for blocking cancer cell proliferation by XPO1 inhibition as well as a novel PML- and p62-mediated mechanism of p53 activation in some types of cancer cells.
Highlights
The abnormal proliferation of cancer cells is driven by deregulated oncogenes or tumor suppressors, among which the cancer-vulnerable genes are attractive therapeutic targets
By performing a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 screening of cells transformed by an oncogenic virus, namely, Kaposi’s sarcoma-associated herpesvirus (KSHV), we have identified cellular genes that are essential for cellular transformation [13]
We have previously shown that infection by KSHV alone is sufficient to efficiently infect and transform mesenchymal stem (MM) cells without depending on cellular genetic alterations and that KSHV-transformed KSHV-transformed primary rat mesenchymal embryonic stem (KMM) cells can efficiently induce tumors in nude mice with pathological features resembling Kaposi’s sarcoma (KS) [13]
Summary
The abnormal proliferation of cancer cells is driven by deregulated oncogenes or tumor suppressors, among which the cancer-vulnerable genes are attractive therapeutic targets. XPO1 inhibition induces relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies These results illustrate that XPO1 is a vulnerable target of cancer cells and reveal a novel mechanism for blocking cancer cell proliferation by XPO1 inhibition as well as a novel PML- and p62-mediated mechanism of p53 activation in some types of cancer cells. Numerous proteins involved in cancer, including p53, adenomatous polyposis coli (APC), retinoblastoma (Rb), NFAT (nuclear factor of activated T cells), and -catenin, are abnormally localized in cancer cells The consequence of these dysfunctions is either overactivation of oncogenes or inactivation of tumor suppressor proteins, resulting in uncontrolled cell proliferation and growth transformation [2]. While importins bind to their cargo proteins in cytoplasm and release them in nucleus upon binding of RanGTP, exportins bind to cargos in nucleus only in the presence of RanGTP and release them in cytoplasm upon Ran-driven GTP hydrolysis [4]
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